22 



removal of the t-BOC group from ADBH by dissolving 6mg of 

 ADBH in 5ml of dry trif luoroacetic acid at room temperature, 

 swirling for 1 minute followed by immediate evaporation of 

 the acid at 40°C in vacuo . The resulting compound, a-amani- 

 tin-diazobenzoylhexamethylenediamine (ADH) , contains a free 

 amino group that may be coupled directly to free protein 

 carboxyl groups by reaction with water soluble carbodiimides 

 (Carraway and Koshland, 1972). Twenty milligrams BSA dis- 

 solved in 2ml water with 200mg of N-ethyl-N' (dimethylamino- 

 propyl) -carbodiimide HC1 (EDC) were added to the dried ADH 

 in a round bottom flask. After 24 hours at room temperature 

 with occasional mixing, the reaction mixture was adjusted to 

 0.05% with respect to NH HCO by addition of solid NH.HCO^, 

 applied to a Sephadex G-75 column (2.5 x 30cm) pre-equili- 

 brated with 0.05% NH .HCO.,, and eluted with the same buffer. 

 Conjugated ADH-BSA eluted as a single peak followed by 

 unreacted ADH which was pooled and concentrated for reuse. 



The peak of protein conjugate was analyzed for protein con- 



1% 

 tent by uv absorbance at 280nm (E ° = 6.61) and by the 

 J 1cm J 



Lowry method (Lowry et al., 1951), for a-amanitin content 

 as indicated from absorption of the diazo linkage at 384nm 

 (E = 14000cm /mMole) and by inhibition of calf thymus 

 RNA polymerase II. After analysis, individual fractions 

 were pooled, lyophilized and stored at -20°C. 

 ADH-Con A 



Conjugates of a-amanitin and concanavalin A (Con A) 

 were prepared in a similar fashion to the ADH-BSA conjugates. 



